Human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), is estimated to affect 36.7 million people worldwide. Approximately 2 million people worldwide become newly infected with HIV each year. HIV rapidly evolves by altering expression of certain viral components making HIV difficult to treat, and to date, nearly impossible to cure. The current standard of treatment uses a combinatorial approach referred to as anti-HIV therapy (ART). While ART reduces overall levels of the HIV in a patient, ART is not curative. Patients undergoing ART endure high costs for treatment and are at risk for suffering from side effects associated with this lifelong therapy.
Gene therapy, a treatment successfully used for other diseases, has been studied as an alternative treatment to ART. One of the major challenges in treating HIV with gene therapy involves its rapidly evolving genome. By altering gene sequences, HIV evades certain treatments to become drug resistant. Early attempts of treating HIV using combinatorial gene therapy included both RNA and protein based strategies that were effective in cell culture and animal models (Li, et al., 2005; Asparuhova, et al., 2008; Anderson, et al., 2009; Chung, et al., 2014). However, these strategies may not be sufficient to reduce HIV, or cure HIV/AIDS, in a patient.
Ribonucleic acid (RNA) aptamers (RNA aptamers and aptamers) are short, single-stranded RNA molecules that fold into stable three-dimensional shapes and are useful for binding to certain structural features of target molecules. RNA aptamers having high affinity and specificity for target molecules, such as proteins, have previously been selected from complex libraries using the Selective Enrichment of Ligands by Exponential Enrichment (SELEX) protocol (Tuerk and Gold, 1990; Ellington and Szostak, 1992). Most therapeutic RNA aptamers are exogenously administered to cells that express a target molecule (e.g., a target cell) by binding to extracellular domains of certain cell surface proteins. These have been used to inhibit a function of the target molecule or as vehicles to deliver a therapeutic agent to the target cell. RNA aptamers having high binding affinity for some HIV specific molecules (e.g., reverse transcriptase (RT or Rev), glycoprotein120 (gp120), group specific antigen (Gag) and protease) have also been identified and isolated (Zhou, et al., 2009; Famalingam, et al., 2011; Ditzler, et al., 2011; Whatley, et al., 2013; Shum, Zhou and Rossi, 2013; Duclair, et al., 2015).
Traditionally, RNA aptamers are transcribed from a DNA polymerase III (Pol III) promoter, such as a U6 promoter, and include defined start and termination sites. This allows for precise prediction of the length and structure of the expressed RNA aptamers. However, when expressed from the Pol III promoter, RNA transcripts corresponding to the aptamers lack an intrinsic nuclear export signal (e.g., a 5′ cap and a polyA tail). As such, intrinsic Pol III transcripts are present in the nucleus, rather than the cytoplasm. While the nuclear export signal can be added to the DNA sequence encoding the aptamer, the signal may alter the structure of the aptamer and interfere with the aptamer's desired function, such as binding to certain structural features of target molecules. In this way, nuclear export signals may not be useful for certain RNA aptamers. As such, intrinsic Pol III transcripts have limited use for RNA aptamers designed to target cytoplasmic proteins. Accordingly, expression of functional aptamers in HIV-infected cells remains a major hurdle for successful application of gene therapy, having long-term stability, to HIV patients.
Thus, it is of importance to develop alternative methods for expressing functional target-specific aptamers that may be applied to a successful combinatorial anti-HIV gene therapy therapeutic approach.